FIG. 8.

Inhibition of autophagy induces stress and apoptosis in β-cells and abolishes the beneficial effects of rapamycin in Akita β-cells. Wild-type and Akita β-cells were treated without and with rapamycin (50 nmol/L) and the lysosomal enzyme inhibitor chloroquine (50 μmol/L) for 16 h, followed by analysis for different markers of ER stress and apoptosis by Western blot (A). S6 phosphorylation was used as a marker for mTORC1 inhibition by rapamycin. Accumulation of LC3-ΙΙ in response to treatment with chloroquine indicates inhibition of autophagic flux (B). JNK phosphorylation, CHOP expression, and cleaved caspase 3 were used to assess stress and apoptosis (C). Quantification of apoptosis was performed by ELISA for cytosolic oligonucleosomes (D). Representative blots and quantifications are shown. Results are means ± SE of three to eight individual experiments. *P < 0.05, **P < 0.01, #P < 0.001 for the difference between the indicated groups. Cl., cleaved; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.